Cells of mesodermal origin with hematopoietic potential appear at distinct times and locations during embryonic development. It is now generally accepted that the extra-embryonic yolk sac and the paraaortic splanchnopleura / aorta-gonad-mesonephros regions of the embryo represent independent sites of hematopoietic development. Most studies indicate that hematopoietic progenitors from these two locations display differential potential in their ability to produce the various hematopoietic lineages and in their capacity to give rise to the long-term repopulating stem cell (LTRSC). These two sites could therefore contain different populations with unique functions during development. The overall goals of the experiments in this proposal are to map the mesodermal origins of these hematopoietic cell populations with the aim of generating the LTRSC from embryonic stern (ES) cells differentiated in culture, The studies proposed here build on extensive analysis and understanding of the early hematopoietic commitment steps in the in vitro ES differentiation model. ES cells that express GFP from the mesodermal gene brachyury locus will be used as a tool to isolate mesodermal populations from in vitro differentiated ES cells. The ability to generate the LTRSC from ES cells will establish a powerful system for the study of stem cell development, which can be used to treat many diseases. The specific aims are: Aim 1. Do Different subpopulations of mesoderm give rise to different hematopoietic programs? Aim 2. Does a specific subpopulation of mesoderm give rise to LTRSCs?